Molecular Mechanisms of TGF-beta Signaling Pathway

通过其膜结合的I型受体的作用，转化生长因子-β引起广泛的细胞反应，调节细胞的增殖、分化和凋亡。许多这些信号反应都是由Smad蛋白介导的。因此，控制Smad的活性对于转化生长因子-β及其相关因子的正确信号传导至关重要。我们发现，除了两个C末端残基外，转化生长因子-β还能诱导Smad3连接区的三个位点的磷酸化，而GSK3负责其中一个位点的磷酸化，即Ser204。在体内GSK3活性的启动位点Ser204和/或邻近的Ser208上的丙氨酸替换增强了Smad3与CBP的亲和力，表明连接蛋白的磷酸化可能是调节Smad3转录活性的负反馈环的一部分。因此，我们的发现揭示了SMAD3信号机制的一个新方面，该机制控制着细胞对转化生长因子-β的最终反应幅度。除了磷酸化，Smad蛋白还受到泛素介导的降解。含有Hect结构域的泛素连接酶SMurf1和SMurf2能够直接与BMP途径的Smad1和Smad5以及转化生长因子-β途径的Smad2和Smad3相互作用，调节其活性。为了阐明SmRf在转化生长因子β信号转导中的生理意义，我们建立了缺失Smrf1或SmRf2的小鼠。虽然Smads参与了转化生长因子-β超家族的大部分活动，但激活的转化生长因子-β受体也通过其他细胞内信号转导途径传递信号，特别是由MAP激酶介导的信号转导。我的团队的第二个研究领域集中在转化生长因子-β受体激活不依赖于Smads的MAP激酶的具体机制，以及这一非Smad依赖的途径在转化生长因子-β信号转导中的生物学意义。为此，我们发现TRAF6是JNK和p38 Smad非依赖性激活所必需的，其羧基TRAF同源结构域与转化生长因子-β受体发生物理作用。转化生长因子-β诱导K63连接的TRAF6泛素化，并促进TRAF6和TAK1之间的关联。我们的结果表明，转化生长因子-β激活JNK和p38的机制与IL-1β/Toll样受体途径类似。目前，我们试图通过鉴定与转化生长因子-βI型受体特异性相关的蛋白质并表征它们的功能来扩展这一发现，以表征Smad非依赖性通路的分子机制。我的小组的第三个方向集中在异常的Smad信号在肿瘤发生中的作用。在四环素抑制启动子(tet-off)的控制下，我们已经产生了不同的转基因小鼠系，携带野生型、显性阴性或Smad3。我们将这些小鼠与LAP-TTA小鼠杂交，LAP-TTA小鼠允许四环素调控的四环素反式激活蛋白(TTA)在肝细胞中特异性表达Smad3及其变异体。我们发现，Smad3的高表达保护肝脏免受化学诱导的癌变，这是由于肝脏对凋亡刺激的反应增强所致。我们计划继续使用这个模型来进一步探索Smad3在肝脏肿瘤进展和转移的晚期阶段中的作用。此外，我们还对Smad信号如何与其他途径融合以及这些信号通路在控制转化生长因子-β调控的基因转录、细胞增殖、分化、凋亡和肿瘤进展中发挥的作用感兴趣。

Through the action of its membrane bound type I receptor, TGF-beta elicits a wide range of cellular responses that regulate cell proliferation, differentiation and apoptosis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-beta and its related factors. We found that TGF-beta induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and GSK3 is responsible for phosphorylation at one of these sites, namely Ser204. Alanine substitution at Ser204 and/or the neighboring Ser208, the priming site for GSK3 in vivo activity, strengthened the affinity of Smad3 to CBP, suggesting that the linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-beta. In addition to phosphorylation, Smad proteins are also subjected to ubiquitin-mediated degradation. The HECT domain-containing ubiquitin ligase Smurf1 and Smurf2 have the ability to interact directly with Smad1 and Smad5 of the BMP pathway and Smad2 and Smad3 of TGF-beta pathway to regulate their activities. To address the physiological significance of Smurfs in TGF-beta signaling, we have generated mice lacking either Smurf1 or Smurf2.Although Smads are involved in most actions of the TGF-beta superfamily, activated TGF-beta receptors also transduce signals through other intracellular signaling pathways, especially those mediated by MAP kinases. The second area of research of my group focuses on the specific mechanism by which TGF-beta receptors activate MAP kinases independent of Smads, and the biological significance of this non-Smad dependent pathway in TGF-beta signaling. Toward this goal, we found that TRAF6 is specifically required for the Smad-independent activation of JNK and p38 and its carboxyl TRAF homology domain physically interacts with TGF-beta receptors. TGF-beta induces K63-linked ubiquitination of TRAF6, and promotes association between TRAF6 and TAK1. Our results indicate that TGF-beta activates JNK and p38 through a mechanism similar to that operating in the interleukin-1beta/Toll-like receptor pathway. Currently, we seek to expand this findings to characterize molecular mechanisms of the Smad-independent pathways by identifying proteins that are specifically associated with TGF-beta type I receptor and characterizing their functions. The third direction of my group focuses on the effect of aberrant Smad signaling in tumorigenesis. We have generated different lines of transgenic mice carrying either wild type, or dominant negative or Smad3 under the control of a tetracycline-repressible promoter (tet-off). We crossed these mice to LAP-tTA mice, which allow tetracycline-regulated expression of tetracycline-transactivating protein (tTA) specifically in hepatocytes, to express Smad3 and its variants in liver. We find that elevated Smad3 expression protects liver from chemically induced carcinogenesis due to a heightened hepatic response to apoptotic stimuli. We plan to continue using this model to further explore the role of Smad3 in late stages of liver tumor progression and metastasis. In addition, we are also interested in how Smad signaling converges with other pathways and what kind of roles these cross-talks play in controlling TGF-beta-regulated gene transcription, cell proliferation, differenciation, apoptosis and tumor progression.